The overall objective is to investigate the molecular and genetic mechanisms of cellular repair of damaged nuclear and mitochondrial DNA and the effect of cellular repair mechanisms on the induction of nuclear and mitochondrial gene mutations. These problems are being studied in a simple eucaryotic organism, the yeast Saccharomyces cerevisiae, where both mutants with repair deficiencies and a well-defined mutational system exist. For the study of nuclear gene mutations, reversion of various mutants of iso-l-cytochrome c are being measured. The main advantage of this system is that the nature of the lesions in the DNA of the mutants can be deduced from the alterations of the primary structure of iso-l-cytochrome c. We measure mutations of mitochondrial genes by the induction of cytoplasmic petites and erythromycin resistance. Reversion frequencies obtained in both repair-deficient and repair- proficient strains show what influence repair capacity has on the induction of nuclear and mitochondrial genes by physical agents such as ionizing radiation and ultraviolet light (UV) and by numerous chemical mutagens and carcinogens. How radiation damage and damage induced by alkylating agents are repaired in yeast also will be investigated by examining altered DNA. Does yeast, a relatively simple eucaryotic microorganism, respond like other eucaryotes by excising monofunctional alkylation products from DNA, or does it behave like procaryotes whose repair systems may have less recognition for alkylated DNA? Does the same repair process act on both nuclear and mitochondrial DNA? Or might nuclear DNA be repaired according to the eucaryotic mode and mitochondrial DNA like procaryotic repair, in view of the bacteria-like nature of mitochondria? This question will be answered by measuring excision of alkylation products from nuclear and mitochondrial DNA. Repair of UV-induced damage in nuclear and mitochondrial DNA will be measured by determining the susceptibility of dimer-containing DNA to the production of single strand breaks after treatment with T4 UV endonuclease. Lastly, we would like to know whether the same or different genes control repair of both types of DNA.